Telomeres, the natural ends of linear eukaryotic chromosomes are specialized protein- DNA complexes that play essential roles in cell viability and genome stability. Gene integrity is critical to keep cells from tumorigenesis. Tumor karyotypes display three main types of alterations: loss of material, gene amplification, and chromosome rearrangement. Each of these changes could result from loss of telomere function in tumor cells. The long-term goal of my research is to understand the molecular mechanisms of how the human telomere executes its two main functions: to protect chromosome ends and mediate their replication. An improved understanding of the structure of human telomeres and how telomere protection and regulation differs between normal cells and cancer cells is critical for preventing, diagnosing, and treating cancer. During the last decade, much information has been accumulated about the protein components of human telomeres. A six-protein complex, called shelterin, is thought to protect the telomeres of human chromosomes. TRF1 and TRF2 directly bind double-stranded telomeric DNA, POT1 directly binds the single-stranded 3'-extension at the chromosome end, and these are bridged through protein-protein interactions involving TIN2 and TPP1. The sixth protein, RAP1, binds mostly to TRF2. The shelterin complex associates with telomeric DNA and forms a key protective structure that is essential for telomere integrity and length regulation. Alterations in this structure correlate with the onset of cellular senescence in normal cells and tumor aggressiveness in cancerous cells. However, the molecular architecture and the regulation of the shelterin complex are still poorly understood. The objective of the proposed research is to determine the three-dimensional structure of the shelterin complex and to elucidate the mechanisms by which it organizes, protects and regulates human telomeres. In Specific Aim 1, we will determine the shelterin structure at the single-stranded region of the telomeres and elucidate how the POT1-TPP1 complex protects human chromosome overhangs preventing unwanted events which could be catastrophic for the genome. In Specific Aim 2, we will characterize the detailed protein-protein interactions of the shelterin complex at the double-stranded region of the telomeres using both biochemical and structural approaches. The results of this work will help us understand how the shelterin complex is organized at the telomeres. TRF1 and TRF2 not only bind the shelterin proteins, but also interact with many other protein factors, especially those involved in the DNA repair pathways. In Specific Aim 3, we will characterize the interactions mediated by TRF1 and TRF2. In addition, we will also identify the optimal TRF1- and TRF2-binding motifs by random peptide screening method, which will help us identify novel TRF1 and TRF2 mediated interactions. Overall, the three specific aims address the structural and functional versatility of the shelterin complex from different perspectives. The proposed study should lead to novel insights into mechanisms of telomere end protection by shelterin, and provide us better understanding of pathways that connect loss of shelterin with chromosome instability. In return, this may benefit our understanding of the cause and mechanisms of cancer.